This invention relates to a novel class of phosphonic acid derivatives that are inhibitors of PTP-1B.
Reversible protein tyrosine phosphorylation, coordinated by the action of protein tyrosine kinases (PTKs) that phosphorylate certain tyrosine residues in polypeptides, and protein tyrosine phosphatases (PTPs) that dephosphorylate certain phosphotyrosine residues, is a key mechanism in regulating many cellular activities. It is becoming apparent that the diversity and complexity of the PTPs and PTKs are comparable, and that PTPs are equally important in delivering both positive and negative signals for proper function of cellular machinery. Regulated tyrosine phosphorylation contributes to specific pathways for biological signal transduction, including those associated with cell division, cell survival, apoptosis, proliferation and differentiation. Defects and/or malfunctions in these pathways may underlie certain disease conditions for which effective means for intervention remain elusive, including for example, malignancy, autoimmune disorders, diabetes, obesity, and infection.
The protein tyrosine phosphatase (PTP) family of enzymes consists of more than 100 structurally diverse proteins in vertebrates, including human PTPs that have in common the conserved 250 amino acid PTP catalytic domain, but which display considerable variation in their non-catalytic segments (Charbonneau et al., 1992 Annu. Rev. Cell Biol. 8:463-493; Tonks, 1993 Semin. Cell Biol. 4:373-453; Andersen et al., (2001 Mol. Cell Biol. 21:7117-36). This structural diversity presumably reflects the diversity of physiological roles of individual PTP family members, which in certain cases have been demonstrated to have specific functions in growth, development and differentiation (Desai et al., 1996 Cell 84:599-609; Kishihara et al., 1993 Cell 74:143-156; Perkins et al., 1992 Cell 70:225-236; Pingel et al., 1989 Cell 58:1055-1065; Schultz et al., 1993 Cell 73:1445-1454). The PTP family includes receptor-like and non-transmembrane enzymes that exhibit exquisite substrate specificity in vivo and that are involved in regulating a wide variety of cellular signaling pathways (Andersen et al., 2001 Mol. Cell. Biol. 21:7117; Tonks et al., 2001 Curr. Opin. Cell Biol. 13:182). PTPs thus participate in a variety of physiologic functions, providing a number of opportunities for therapeutic intervention in physiologic processes through alteration (i.e., a statistically significant increase or decrease) or modulation (e.g., up-regulation or down-regulation) of PTP activity.
Although recent studies have also generated considerable information regarding the structure, expression and regulation of PTPs, the nature of many tyrosine phosphorylated substrates through which the PTPs exert their effects remains to be determined. Studies with a limited number of synthetic phosphopeptide substrates have demonstrated some differences in the substrate selectivities of different PTPs (Cho et al., 1993 Protein Sci. 2: 977-984; Dechert et al., 1995 Eur. J Biochem. 231:673-681). Analyses of PTP-mediated dephosphorylation of PTP substrates suggest that catalytic activity may be favored by the presence of certain amino acid residues at specific positions in the substrate polypeptide relative to the phosphorylated tyrosine residue (Salmeen et al., 2000 Molecular Cell 6:1401; Myers et al., 2001 J. Biol. Chem. 276:47771; Myers et al., 1997 Proc. Natl. Acad. Sci. USA 94:9052; Ruzzene et al., 1993 Eur. J. Biochem. 211:289-295; Zhang et al., 1994 Biochemistry 33:2285-2290). Thus, although the physiological relevance of the substrates used in these studies is unclear, PTPs display a certain level of substrate selectivity in vitro.
The PTP family of enzymes contains a common evolutionarily conserved segment of approximately 250 amino acids known as the PTP catalytic domain. Within this conserved domain is a unique signature sequence motif, CX5R, that is invariant among all PTPs. In a majority of PTPs, an 11 amino acid conserved sequence ([I/V]HCXAGXXR[S/T)G (SEQ ID NO:1)) containing the signature sequence motif is found. The cysteine residue in this motif is invariant in members of the family and is essential for catalysis of the phosphotyrosine dephosphorylation reaction. It functions as a nucleophile to attack the phosphate moiety present on a phosphotyrosine residue of the incoming substrate. It is well-known that if the cysteine residue is altered by site-directed mutagenesis to serine (e.g., in cysteine-to-serine or “CS” mutants) or alanine (e.g., cysteine-to-alanine or “CA” mutants), the resulting PTP is catalytically deficient but retains the ability to complex with, or bind, its substrate, at least in vitro.
One non-transmembrane PTP, PTP1B, recognizes several tyrosine-phosphorylated proteins as substrates, many of which are involved in human disease. For example, therapeutic inhibition of PTP1B in the insulin signaling pathway may serve to augment insulin action, thereby ameliorating the state of insulin resistance common in Type II diabetes patients. PTP1B acts as a negative regulator of signaling that is initiated by several growth factor/hormone receptor PTKs, including p210 Bcr-Abl (LaMontagne et al., 1998 Mol. Cell. Biol. 18:2965-75; LaMontagne et al., 1998 Proc. Natl. Acad. Sci. USA 95:14094-99), receptor tyrosine kinases, such as EGF receptor, PDGF receptor, and insulin receptor (IR) (Tonks et al., 2001 Curr. Opin. Cell Biol. 13:182-95), and JAK family members such as Jak2 and others (Myers et al., 2001 J. Biol. Chem. 276:47771-74), as well as signaling events induced by cytokines (Tonks et al., 2001). Activity of PTP1B is regulated by modifications of several amino acid residues, such as phosphorylation of Ser residues (Brautigan et al., 1993; Dadke et al., 2001; Flint et al., 1993), and oxidation of the active Cys residue in its catalytic motif (Lee et al., 1998; Meng et al., 2002) which is evolutionary conserved among protein tyrosine phosphatases and dual phosphatase family members (Andersen et al., 2001). In addition, changes in the expression levels of PTP1B have been noted in several human diseases, particularly those associated with disruption of the normal patterns of tyrosine phosphorylation.
Diabetes mellitus is a common, degenerative disease affecting 5-10% of the human population in developed countries, and in many countries, it may be one of the five leading causes of death. Approximately 2% of the world's population has diabetes, the overwhelming majority of cases (>90%) being type 2 diabetes and the remainder being type 1. In type 1 diabetes, which is frequently diagnosed in children or young adults, insulin production by pancreatic islet beta cells is destroyed. Type 2 diabetes, or “late onset” or “adult onset” diabetes, is a complex metabolic disorder in which cells and tissues cannot effectively use available insulin; in some cases insulin production is also inadequate. At the cellular level, the degenerative phenotype that may be characteristic of late onset diabetes mellitus includes, for example, impaired insulin secretion and decreased insulin sensitivity, i.e., an impaired response to insulin.
Studies have shown that diabetes mellitus may be preceded by or is associated with certain related disorders. For example, an estimated forty million individuals in the U.S. suffer from late onset impaired glucose tolerance (IGT). IGT patients fail to respond to glucose with increased insulin secretion. Each year a small percentage (5-10%) of IGT individuals progress to insulin deficient non-insulin dependent diabetes (NIDDM). Some of these individuals further progress to insulin dependent diabetes mellitus (IDDM). NIDDM and IDDM are associated with decreased release of insulin by pancreatic beta cells and/or a decreased response to insulin by cells and tissues that normally exhibit insulin sensitivity. Other symptoms of diabetes mellitus and conditions that precede or are associated with diabetes mellitus include obesity, vascular pathologies, and various neuropathies, including blindness and deafness.
Type 1 diabetes is treated with lifelong insulin therapy, which is often associated with undesirable side effects such as weight gain and an increased risk of hypoglycemia. Current therapies for type 2 diabetes (NIDDM) include altered diet, exercise therapy, and pharmacological intervention with injected insulin or oral agents that are designed to lower blood glucose levels. Examples of such presently available oral agents include sulfonylureas, biguanides, thiazolidinediones, repaglinide, and acarbose, each of which alters insulin and/or glucose levels. None of the current pharmacological therapies, however, controls the disease over its full course, nor do any of the current therapies correct all of the physiological abnormalities in type 2 NIDDM, such as impaired insulin secretion, insulin resistance, and excessive hepatic glucose output. In addition, treatment failures are common with these agents, such that multi-drug therapy is frequently necessary.
In certain metabolic diseases or disorders, one or more biochemical processes, which may be either anabolic or catabolic (e.g., build-up or breakdown of substances, respectively), are altered (e.g., increased or decreased in a statistically significant manner) or modulated (e.g., up- or down-regulated to a statistically significant degree) relative to the levels at which they occur in a disease-free or normal subject such as an appropriate control individual. The alteration may result from an increase or decrease in a substrate, enzyme, cofactor, or any other component in any biochemical reaction involved in a particular process. Altered (i.e., increased or decreased in a statistically significant manner relative to a normal state) PTP activity can underlie certain disorders and suggests a PTP role in certain metabolic diseases.
For example, disruption of the murine PTP1B gene homolog in a knock-out mouse model results in PTP1B−/− mice exhibiting enhanced insulin sensitivity, decreased levels of circulating insulin and glucose, and resistance to weight gain even on a high-fat diet, relative to control animals having at least one functional PTP1B gene (Elchebly et al., Science 283:1544 (1999)). Insulin receptor hyperphosphorylation has also been detected in certain tissues of PTP1B deficient mice, consistent with a PTP1B contribution to the physiologic regulation of insulin and glucose metabolism (Id.). PTP1B-deficient mice exhibit decreased adiposity (reduced fat cell mass but not fat cell number), increased basal metabolic rate and energy expenditure, and enhanced insulin-stimulated glucose utilization (Klaman et al., 2000 Mol. Cell. Biol. 20:5479). Additionally, altered PTP activity has been correlated with impaired glucose metabolism in other biological systems (e.g., McGuire et al., 1991 Diabetes 40:939; Myerovitch et al., 1989 J. Clin. Invest. 84:976; Sredy et al., 1995 Metabolism 44:1074), including PTP involvement in biological signal transduction via the insulin receptor (see, e.g., WO 99/46268 and references cited therein).
An integration of crystallographic, kinetic, and PTP1B-peptide binding assays illustrated the interaction of PTP1B and insulin receptor (IR) (Salmeen et al., 2000 Mol. Cell 6:1401-12). The insulin receptor (IR) comprises two extracellular α subunits and two transmembrane β subunits. Activation of the receptor results in autophosphorylation of tyrosine residues in both β subunits, each of which contains a protein kinase domain. Extensive interactions that form between PTP1B and insulin receptor kinase (IRK) encompass tandem pTyr residues at 1162 and 1163 of IRK, such that pTyr-1162 is located in the active site of PTP1B (id.). The Asp/Glu-pTyr-pTyr-Arg/Lys motif has been implicated for optimal recognition by PTP1B for IRK. This motif is also present in other receptor PTKs, including Trk, FGFR, and Axl. In addition, this motif is found in the JAK family of PTKs, members of which transmit signals from cytokine receptors, including a classic cytokine receptor that is recognized by the satiety hormone leptin (Touw et al., 2000 Mol. Cell. Endocrinol. 160:1-9).
Changes in the expression levels of PTP1B have been observed in several human diseases, particularly in diseases associated with disruption of the normal patterns of tyrosine phosphorylation. For example, the expression of PTP1B is induced specifically by the p210 Bcr-Abl oncoprotein, a PTK that is directly responsible for the initial manifestations of chronic myelogenous leukemia (CML) (LaMontagne et al., 1998 Mol. Cell. Biol. 18:2965-75; LaMontagne et al., 1998 Proc. Natl. Acad. Sci. USA 95:14094-99). Expression of PTPB1 in response to this oncoprotein is regulated, in part, by transcription factors Sp1, Sp3, and Egr-1 (Fukada et al., 2001 J. Biol. Chem. 276:25512-19). These transcription factors have been shown to bind to a p210 Bcr-Abl responsive sequence (PRS) in the human PTP1B promoter, located between −49 to −37 base pairs from the transcription start site, but do not appear to mediate certain additional, independent PTP1B transcriptional events, for which neither transcription factor(s) nor transcription factor recognition element(s) have been defined (id.).
Another protein tyrosine phosphatase enzyme, T-cell protein tyrosine phosphatase, or TCPTP, dephosphorylates JAK1 and JAK3. TCPTP appears to have a role in the regulation of immune homeostasis. TCPTP is also known to dephosphorylate the EGF receptor, and the adapter protein p52shc. TCPTP may also play a role in certain T-cell malignancies. (See, for example, Simoncic et al., Curr. Biol. Mar. 19, 2002; 12(6):446-45; Heinonen et al., 2004 Blood, 103:3457-3464; You-Ten et al., 1997 J. Exp. Med., 186:683-693). TCPTP also may play a role in insulin-signaling. (See Tonks et al., 2004 JBC, 279:37716; and 2003 Mol. Cell Biol. 23:2096).
Currently, therefore, desirable goals for therapeutic regulation of biological signal transduction include modulation of PTP1B-mediated cellular events including, inter alia, inhibition or potentiation of interactions among PTP1B-binding molecules, substrates and binding partners, or of other agents that regulate PTP1B activities. Accordingly, a need exists in the art for an improved ability to intervene in the regulation of phosphotyrosine signaling, including regulating PTP1B by altering PTP1B catalytic activity, PTP1B binding to PTP1B substrate molecules, and/or PTP1B-encoding gene expression. An increased ability to so regulate PTP1B may facilitate the development of methods for modulating the activity of proteins involved in phosphotyrosine signaling pathways and for treating conditions associated with such pathways. The present invention fulfills these needs and further provides other related advantages.